WO2022142714A1

WO2022142714A1 - Use of rutaecarpine in inhibition of cell migration

Info

Publication number: WO2022142714A1
Application number: PCT/CN2021/128385
Authority: WO
Inventors: 赵斌; 刘奕耘; 谢群慧; 徐丽; 陈旸升
Original assignee: 中国科学院生态环境研究中心
Priority date: 2020-12-31
Filing date: 2021-11-03
Publication date: 2022-07-07
Also published as: CN116916926A

Abstract

Provided is the use of rutaecarpine in the inhibition of cell migration, in particular the use of rutaecarpine or a pharmaceutical composition containing rutaecarpine in the preparation of a drug for inhibiting the migration of glioma cells. It is found for the first time that a specific concentration of rutaecarpine can inhibit the migration of glioblastoma cell line U87, thus providing a new choice for the treatment of glioblastomas.

Description

Application of Evodiamine in Inhibiting Cell Migration

The present application is based on the CN application number 202011630864.3 and the filing date is December 31, 2020, and claims its priority. The disclosure of the CN application is hereby incorporated into the present application as a whole.

technical field

The present application relates to technologies related to cancer treatment, in particular, the present application relates to the application of evodiamine in inhibiting cell migration, and particularly discloses the application of evodiamine in inhibiting glioblastoma cell migration.

Background technique

Brain and central nervous system tumors are the third leading cause of cancer-related death among all cancer-related case studies. Among brain tumors, astrocytoma has a higher incidence, accounting for 15.1% of primary central nervous system tumors and 46.1% of primary central nervous system malignancies. Among them, glioblastoma is the most common and belongs to a very aggressive primary glioma, which is also a grade IV tumor in the World Health Organization (WHO) classification of tumor malignancy.

The comprehensive treatment of glioblastoma includes surgery, radiotherapy, systemic therapy (chemotherapy, targeted therapy) and supportive care, but the overall prognosis is still poor and the long-term survival rate is very low. There is still a need in the art for more therapeutic approaches for glioblastoma.

SUMMARY OF THE INVENTION

Studies have shown diffuse growth of glioblastoma, and in addition to short-distance intracranial migration, extracranial metastasis of tumor cells has also been found. Therefore, inhibiting the migratory movement of glioblastoma may be beneficial for tumor therapy, reducing the risk of postoperative recurrence and improving disease prognosis.

Rutaecarpine is extracted from the fruit of the Rutaceae plant, Evodia rutaecarpa (Juss.) Benth, and the structure is shown below. Research results show that it has anti-inflammatory and cardiovascular protection effects, but there is no relevant report on the inhibition of glioblastoma by evodiamine.

It is found in the present application that evodial can effectively inhibit the migration of glioblastoma, and has low cytotoxicity, so it can be an effective therapeutic drug for glioblastoma.

Therefore, in one aspect, the present invention provides the use of evodiamine or a pharmaceutical composition containing evodiamine in the preparation of a medicament for inhibiting glioma cell migration.

In some embodiments, the glioma cells are astrocytoma cells.

In some embodiments, the glioma cells are glioblastoma cells.

In some embodiments, the glioblastoma cells are cells or glioblastoma cell lines from a subject.

In some embodiments, the subject has glioblastoma.

In some embodiments, the glioma cell line is the glioblastoma cell line U87.

In some embodiments, the concentration of evodiamine is ^10-7 to ^10-4 M, such as 1× ^10-7 to 1× ^10-6 M, 1× ^10-7 to 1× ^10-5 M, 1× ^10-7 to 1× ^10-4 M, 1× ^10-6 to 1× ^10-5 M, 1× ^10-6 to 1× ^10-4 M, or 1× ^10-5 to 1× ^10-4 M .

In another aspect, the present invention provides the use of evodiamine or a pharmaceutical composition containing evodiamine in the preparation of a medicament for preventing and/or treating glioma.

In some embodiments, the medicament is for preventing and/or inhibiting glioma growth, metastasis and/or invasion.

In some embodiments, the glioma is an astrocytoma.

In some embodiments, the glioma is a glioblastoma.

In some embodiments, the pharmaceutical composition also contains a pharmaceutically acceptable carrier and optionally other therapeutic agents.

In some embodiments, the additional therapeutic agent is selected from the group consisting of temozolomide, bevacizumab, lomustine, carmustine, carboplatin, procarbazine, and vincristine.

In another aspect, the present invention provides a method of inhibiting glioma cell migration, comprising the step of contacting the cells with an effective amount of evodiamine or a pharmaceutical composition containing evodiamine.

In some embodiments, the glioma cells are astrocytoma cells.

In some embodiments, the glioma cells are glioblastoma cells.

In some embodiments, the subject has glioblastoma.

In some embodiments, the glioma cell line is the glioblastoma cell line U87.

In some embodiments, the concentration of evodiamine is ^10-7 to ^10-4 M, such as ^10-7 M to ^10-6 M, ^10-7 M to ^10-5 M, ^10-7 M to ^10-4 M, 10 ^-6 M to 10 ^-5 M, 10 ^-6 M to 10 ^-4 M, or 10 ^-5 M to 10 ^-4 M.

In some embodiments, the method is performed in vivo.

In some embodiments, the method is performed in vitro.

In another aspect, the present invention provides a method of preventing and/or treating glioma, comprising administering to a subject in need thereof a prophylactically and/or therapeutically effective amount of evodiamine or a pharmaceutical combination containing evodiamine the steps of the thing.

In some embodiments, the methods are for preventing and/or inhibiting glioma growth, metastasis and/or invasion.

In some embodiments, the glioma is an astrocytoma.

In some embodiments, the glioma is a glioblastoma.

In some embodiments, evodiamine alone or a pharmaceutical composition containing evodiamine is administered; or,

Simultaneous, sequential, or sequential administration of evodiamine or a pharmaceutical composition containing evodiamine, and other therapeutic agents. In some preferred embodiments, the additional therapeutic agent is selected from the group consisting of temozolomide, bevacizumab, lomustine, carmustine, carboplatin, procarbazine, and vincristine.

In another aspect, the present application provides evodiamine or a pharmaceutical composition containing evodiamine for use in inhibiting glioma cell migration.

In some embodiments, the glioma cells are astrocytoma cells.

In some embodiments, the glioma cells are glioblastoma cells.

In some embodiments, the subject has glioblastoma.

In some embodiments, the glioma cell line is the glioblastoma cell line U87.

In some embodiments, the concentration of evodiamine is 1× ^10-7 to 1× ^10-4 M, such as 1× ^10-7 to 1× ^10-6 M, 1× ^10-7 to 1× ^10-5 M, 1×10 ^-6 to 1×10 ^-5 M, 1×10 ^-6 to 1×10 ^-4 M, or 1×10 ^-5 to 1×10 ^-4 M.

In another aspect, the present application provides evodiamine or a pharmaceutical composition containing evodiamine for use in the treatment of gliomas.

In some embodiments, the evodiamine or a pharmaceutical composition containing evodiamine is used to inhibit glioma growth, metastasis and/or invasion.

In some embodiments, the glioma is an astrocytoma; preferably, the glioma is a glioblastoma.

In some embodiments, the pharmaceutical composition further contains a pharmaceutically acceptable carrier and optionally other therapeutic agents; preferably, the other therapeutic agents are selected from temozolomide, bevacizumab, lomustine, cardinolide mustine, carboplatin, procarbazine, and vincristine.

Definition of Terms

Molecular biology, microbiology, and recombinant DNA techniques that may be used in this application are those known in the art. These techniques are fully explained in the literature. See, e.g.: Sambrook, Fritsch & Maniatis, Molecular Cloing: A Laboratory Mannual, (1982); DNA Cloning: A Practical Approach Volumes I&II, D.N. Glover ed. 1985; Oligonucleotide Synthesis, M.J.Gait ed.1984; Nucleic Acid Hybridization, B.D. Hames&S .J.Higgins eds.1985; Transcription and Translation, B.D.Hames & S.J.Higgins eds.1984; Animal Cell Culture, R.I.Freshney, ed.1986; Immobilized Cells And Enzymes, IRL Press, 1986; BPerbal, A Practical Guide To Molecular Cloning, 1984. Based on this, terms appearing in this paper are defined as follows.

As used in the specification and the appended claims, the singular forms "a/an" and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a molecule" optionally includes a combination of two or more such molecules, and the like.

As used herein, the term "pharmaceutically acceptable carrier" refers to ingredients other than the active ingredient in a pharmaceutical formulation that are not toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers or preservatives, and the like.

As used herein, the term "individual" or "subject" is a mammal, such as a bovine, equine, porcine, canine, feline, rodent, primate; wherein, Particularly preferred subjects are humans.

As used herein, the term "effective amount" refers to an amount sufficient to obtain, or at least partially obtain, the desired effect. For example, a therapeutically effective amount refers to an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease. Determining such effective amounts is well within the ability of those skilled in the art. For example, an amount effective for therapeutic use will depend on the severity of the disease to be treated, the general state of the patient's own immune system, the patient's general condition such as age, weight and sex, the mode of administration of the drug, and other concurrently administered treatments and many more.

The amount of drug administered to a subject depends on the type and severity of the disease or condition and on the characteristics of the subject, such as general health, age, sex, weight, and tolerance to the drug, and on the formulation The type of drug and how the drug is administered, as well as factors such as the dosing cycle or time interval. Those skilled in the art will be able to determine appropriate dosages based on these and other factors.

As used herein, the terms "glioma", "glioma" refer to tumors of neuroepithelial origin, including primarily astrocytoma, oligodendroglioma, ependymoma by pathological classification And anaplastic (malignant) ependymoma, mixed glioma and choroid plexus. Glioblastoma is the most malignant glioma among astrocytomas.

As used herein, the term "metastasis" is an area of cancer cells that is different from the location of the primary tumor caused by the spread of cancer cells from the primary tumor to other parts of the body. In diagnosing a primary tumor mass, a subject can be monitored for the presence of metastases. Metastases are most often diagnosed by the use of magnetic resonance imaging (MRI) scans, computed tomography (CT) scans, blood and platelet counts, liver function studies, chest X-rays and bone scans, alone or in combination, and for specific symptoms. monitoring to detect. Tumor metastatic behavior may involve several stages, namely: (a) detachment of cells from the tumor; (b) movement of detached cells into surrounding tissue; (c) movement through surrounding tissue into the circulatory system; and (d) movement into circulation system (cells can eventually leave the circulatory system to form secondary tumors). Inhibiting or reducing cell viability in any one or more of these stages will therefore help to at least reduce metastasis. The effect of the drug on each of these subphases can be determined by a number of experiments, namely: (a) the effect of the test drug on cell adhesion; (b) the effect of the test drug on cell lateral movement; (c) the effect of the test drug on cell adhesion. effect of lateral migration of cells; and (d) the effect of the test drug on cell invasiveness, ie the ability of cells to move through the medium that is consumed by the cells. Experiments conducted in connection with the present application show that administration of evodiamine at different dose levels can enhance cell adhesion and/or reduce one or more of lateral movement, lateral migration and invasiveness of cells.

As used herein, invasion of cancer cells refers to the proliferation of cancer cells by penetrating surrounding tissue at the site where the cancer originally occurred, and the direct migration of cancer cells to and penetration of adjacent tissues.

Description of drawings

The accompanying drawings described herein are used to provide a further understanding of the present invention and constitute a part of the present application. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the attached image:

Figure 1 shows the changes of absorbance values of U87 cells detected by microplate reader at 24h and 48h after 10 ^-7 ~ 10 ^-4 M evodial alkaloid treatment.

Figure 2 shows the results of the scratch test to evaluate the effect of evodiamine on the migration ability of U87 cells. Among them, Fig. 2A shows that after 10 ^-5 M evodial alkaloid treatment of cells for 24h, the area of intercellular scratches is larger than that of the control group, indicating that the migration distance of cells is reduced, and the migration ability of cells is decreased; Fig. 2B shows that after 10 ^-5 M evodiola The migration distance of U87 cells was reduced by 23.1% after treatment with subbase, indicating that Evodiamine can effectively inhibit cell migration.

Figure 3 shows the inhibition of glioblastoma cell growth in a mouse tumor model by evodial. Within the first 10 days of administration, the tumor growth rates of the four treatment groups were similar. From the 10th day onward, the growth of tumor volume in the antitumor drug semustine-treated group (positive control group, PC) decreased significantly, followed by the high-dose evodiamine-treated group and the low-dose evodiamine-treated group. By the 21st day of administration, the tumor volume of the high-dose evodial and PC group was significantly smaller than that of the sodium carboxymethylcellulose solvent-treated group (negative control group, NC). It is shown that evodiamine can inhibit the growth of glioblastoma tumor in animal model.

Detailed ways

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, but not all of the embodiments. The following description of at least one exemplary embodiment is merely illustrative in nature and is in no way intended to limit the invention, its application, or uses in any way. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

U87 cells were purchased from the Cell Resource Center, Chinese Academy of Medical Sciences.

Evodimine was purchased from Solarbio Company (Beijing).

The CCK8 kit was purchased from Sangon Biotech (Shanghai).

Microscope: CKX41 (Olympus Japan)

Camera: 600D (Canon Japan)

Microplate reader: Tecan 2000pro (Tecan Switzerland)

Example 1 Study on the toxic effect of evodiamine on glioblastoma

U87 cells were seeded into 96-well plates at a concentration of 1×10 ⁵ cells/mL, with 100 mL of cell suspension per well. Incubate overnight to wait for cells to adhere. Remove the 96-well plate, aspirate the original medium, and wash once with phosphate buffered saline (PBS). The experiment consisted of a background control group, a solvent control group and a drug treatment group. Correspondingly, low-serum medium (serum concentration of 1%, no cells in background control wells), low-serum medium containing solvent dimethyl sulfoxide (DMSO) or different concentrations of evodiamine was added to each well to maintain the solvent. The volume is 0.1%. Put the 96-well plate after dosing into the incubator and continue to cultivate for 24h or 48h. After reaching the culture end time, add 10 μL of CCK8 reagent to each well according to the instructions of the kit, and put the well plate into the incubator again for 1 hour, and then read the absorbance value with a microplate reader.

It can be seen from Figure 1 that the treatment of U87 cells with 10 ^-7 -10 ^-4 M evodiamine within 24h did not cause significant changes in absorbance, and had no effect on cell proliferation and viability. At 48h, although 10 ^-5 M and 10 ^-4 M concentrations of Evodiamine slightly inhibited cell viability, the degree of inhibition was small. Therefore, Evodiamine at this concentration and treatment time had almost no cytotoxicity and could be used in subsequent experiments. usage of.

Example 2. Evodiamine inhibits the migration of glioblastoma cells

The migratory ability of the cells was assessed using a scratch assay. Use a marker to draw 3 straight lines laterally on the bottom of the 6-well plate to locate the scratches. Cells were seeded in 2 mL of 6-well plates at a density of 4×10 ⁵ cells/mL, and cultured overnight until cells adhered. After cells completely cover the bottom of the plate, scratch the cell monolayer with the tip of a 1 mL pipette tip to form a scratch. Select the junction of the scratch and the mark line as the photo location. Floating cells were removed by washing with phosphate buffer, and then low serum medium containing the solvent DMSO or evodial was added. Images were collected under an inverted microscope with a digital camera at 0-24 h after scratching. A total of three independent replicate experiments were performed, each with 3 replicate wells for each concentration group, and each well contained 3 scratches, and 12 images were taken for counting and quantification. Cell migration distance was calculated by dividing the scratch reduction area by the scratch length and analyzed using Image Pro Plus 6.0 software. The scale bar on representative scratch images is 0.5 mm.

As shown in Figure 2A, after the cells were treated with evodial alkaloid for 24 h, the area of the intercellular scratches was larger than that of the control group, indicating that the migration distance of the cells was reduced, and the migration ability of the cells was decreased. According to the statistical data of Figure 2B, the migration distance of U87 cells was reduced by 23.1% after treatment with 10 ^-5 M evodial.

Example 3. Evodiamine inhibits the growth of glioblastoma cells in a mouse tumor model

Twelve male Balb/c nude mice were purchased. Mice had free access to food and water, and the temperature was maintained at about 26 °C, with 12 hours of light and 12 hours of darkness every day. Compounds are exposed as follows:

(1) Take male Balb/c nude mice with a mouse age of 4-6 weeks;

(2) 2×10 ⁶ cells/200 μL of U87 cells were injected subcutaneously in the right axilla of nude mice with a 6-gauge needle, and subcutaneous nodules appeared about 3 days later;

(3) The intragastric administration was started on the third day of the subcutaneous nodule, and the nude mice were randomly divided into 4 groups, with 3 mice in each group;

Negative control group: 0.5% sodium carboxymethyl cellulose 10mL/kg/d

Positive control group: methylcyclonitrosourea (smustine) 5mg/kg

Low-dose group: 10mg/kg/d evodiamine

High-dose group: 20 mg/kg/d evodiamine

Each group was administered with 10 mL of drug/kg body weight of mice

(4) The tumor size was measured every 3 days after administration, and the tumor volume was calculated according to the formula=(long diameter×short diameter2)/2(cm2) to draw the tumor growth curve.

It can be seen from Figure 3 that in the first 10 days of administration, the tumor growth rates of the four treatment groups were almost the same. From the 10th day onward, the growth of tumor volume in the antitumor drug semustine-treated group (positive control group, PC) decreased significantly, followed by the high-dose evodiamine-treated group and the low-dose evodiamine-treated group. By the 21st day of administration, the tumor volume of the high-dose evodial and PC group was significantly smaller than that of the sodium carboxymethylcellulose solvent-treated group (negative control group, NC). It is shown that evodiamine can inhibit the growth of glioblastoma tumor in animal model.

Various modifications of the invention, in addition to those described herein, will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. Each reference cited in this application, including all patents, patent applications, journal articles, books, and any other publications, is incorporated by reference in its entirety.

Claims

Use of evodiamine or a pharmaceutical composition containing evodiamine in the preparation of a medicament for inhibiting glioma cell migration.
The use of claim 1, wherein the glioma cells are astrocytoma cells.
The use of claim 1 or 2, wherein the glioma cells are glioblastoma cells.
The use of claim 3, wherein the glioblastoma cells are cells or glioblastoma cell lines from a subject;

Preferably, the subject has glioblastoma;

Preferably, the glioma cell line is the glioblastoma cell line U87.
The use according to any one of claims 1-4, wherein the concentration of evodiamine is 1 × 10 -7 to 1 × 10 -4 M, such as 1 × 10 -7 to 1 × 10 -6 M, 1 × 10 -7 to 1×10 -5 M, 1×10 -7 to 1×10 -4 M, 1×10 -6 to 1×10 -5 M, 1×10 -6 to 1×10 -4 M, or 1×10 -5 to 1×10 -4 M.
Use of evodiamine or a pharmaceutical composition containing evodiamine in the preparation of a medicament for preventing and/or treating glioma.
The use of claim 6, wherein the medicament is for preventing and/or inhibiting glioma growth, metastasis and/or invasion.
The use of claim 6 or 7, wherein the glioma is an astrocytoma; preferably, the glioma is a glioblastoma.
The use according to any one of claims 1-8, wherein the pharmaceutical composition further contains a pharmaceutically acceptable carrier and optional other therapeutic agents; preferably, the other therapeutic agents are selected from temozolomide, bevacizumab Antibiotics, lomustine, carmustine, carboplatin, procarbazine, and vincristine.
A method of inhibiting the migration of glioma cells, comprising the step of contacting the glioma cells with an effective amount of evodiamine or a pharmaceutical composition containing evodiamine.
The method of claim 10, wherein the glioma cells are astrocytoma cells.
The method of claim 10 or 11, wherein the glioma cells are glioblastoma cells.
The method of claim 12, wherein the glioblastoma cells are cells from a subject or a glioblastoma cell line;

Preferably, the subject has glioblastoma;

Preferably, the glioma cell line is the glioblastoma cell line U87.
The method of any one of claims 10-13, wherein the concentration of evodiamine is 1 × 10 -7 to 1 × 10 -4 M, such as 1 × 10 -7 to 1 × 10 -6 M, 1 × 10 -7 to 1×10 -5 M, 1×10 -6 to 1×10 -5 M, 1×10 -6 to 1×10 -4 M, or 1×10 -5 to 1×10 -4 M.
A method for preventing and/or treating glioma, comprising the step of administering a prophylactically and/or therapeutically effective amount of evodiamine or a pharmaceutical composition containing evodiamine to a subject in need thereof.
The method of claim 15, which is used for preventing and/or inhibiting glioma refers to inhibiting the growth, metastasis and/or invasion of glioma.
The method of claim 15 or 16, wherein the glioma is an astrocytoma; preferably, the glioma is a glioblastoma.
The method of any one of claims 10-17, administering evodiamine alone or a pharmaceutical composition containing evodiamine; or,

Simultaneous, sequential or sequential administration of evodiamine or a pharmaceutical composition containing evodiamine, and other therapeutic agents; preferably, the other therapeutic agents are selected from temozolomide, bevacizumab, lomustine, carmustine , carboplatin, procarbazine, and vincristine.
Evodiamine or a pharmaceutical composition containing evodiamine, which is used for inhibiting glioma cell migration.
The evodiamine or the pharmaceutical composition containing evodiamine according to claim 19, wherein the glioma cells are astrocytoma cells.
The evodiamine or the pharmaceutical composition containing evodiamine according to claim 19 or 20, wherein the glioma cells are glioblastoma cells.
The evodiamine or the pharmaceutical composition containing evodiamine according to claim 21, wherein the glioblastoma cell is a cell or a glioblastoma cell line from a subject;

Preferably, the subject has glioblastoma;

Preferably, the glioma cell line is the glioblastoma cell line U87.
The evodiamine or the pharmaceutical composition containing evodiam according to any one of claims 19-22, wherein the concentration of evodiam is 1×10 -7 to 1×10 -4 M, such as 1×10 -7 ～1× 10-6M , 1× 10-7 ～1× 10-5M , 1× 10-6 ～1× 10-5M , 1× 10-6 ～1× 10-4M , or 1× 10 -5 to 1×10 -4 M.
Evodiamine or a pharmaceutical composition containing evodiamine, which is used for treating glioma.
The evodiamine or the pharmaceutical composition containing evodiamine according to claim 24, which is used for inhibiting the growth, metastasis and/or invasion of glioma.
The evodiamine or the pharmaceutical composition containing evodiamine according to claim 24 or 25, wherein the glioma is an astrocytoma; preferably, the glioma is a glioblastoma.
The evodiamine or the pharmaceutical composition containing evodiamine according to any one of claims 24-26, wherein the pharmaceutical composition also contains a pharmaceutically acceptable carrier and optionally other therapeutic agents; preferably, the Said other therapeutic agent is selected from the group consisting of temozolomide, bevacizumab, lomustine, carmustine, carboplatin, procarbazine, and vincristine.